Myosin I's are widely expressed in different tissues and across broad phylogenetic backgrounds. In intestinal epithelial brush borders, myosin I is localized in the microvillus where it bridges gaps between the membrane and the actin bundles. Myosin I is also localized near the actively ruffling edges of migrating cells. We are using the Baculovirus/Sf9 system to express full-length chicken brush border myosin I heavy chain (BBMI HC) along with calmodulin (CaM), and a truncated head fragment. Vertebrate myosin I's are constitutively active whereas myosin I's from low eukaryotes such as Acanthamoeba and Dictyostelium require phosphorylation at a serine located in the myosin head domain for activity. Sequence alignments of vertebrate and Acanthamoeba myosin I's reveal that the vertebrate proteins have a negatively charged amino acid at the position where the Acanthamoeba protein has the phosphorylatable serine. We intend to explore, using site-directed mutagenesis and the Baculovirus/Sf9 system, whether a negative charge at this site is essential for actin-activated MgATPase activity and in vitro motility.